Nitrate (NO3-) is one of the most common targets for water quality control. High nitrate concentration in the water systems negatively impact human health and the environment. Excessive nitrate content in the aquatic environment led to a decrease in water quality, which can be evaluated from changes in color, odor, increased turbidity, and reduced levels of dissolved oxygen. Denitrification as a biological method of tackling nitrate pollution has been studied since three decades ago in various systems, including recent investigations into bioelectrochemical systems. Bioelectrochemical cells are driven by microorganisms in carrying out oxidation/reduction reactions such as denitrification. However, the popularity of BES is still low. This study aim to optimize the source of carbon as substrate in culture media for the system and compare the efficiency of nitrate elimination and denitrification rate among isolates and culture conditions. Axenic cultures of Pseudomonas entomophila and Acinetobacter oleivorans were done in 20 mL single-chambered Microbial Electrolysis Cells (MECs) equipped with graphite as anode and stainless steel mesh as cathode. Sodium acetate and sodium bicarbonate was used as carbon sources in the formulation of heterotrophic and mixotrophic media. Denitrification performance in bioelectrochemical cells was evaluated from the evolution of nitrate concentration in the medium and the growth of target microorganisms. Environmental isolates presented similar behavior on the medium supplemented with bicarbonate by reaching 68-69% maximum nitrate removal efficiency across all experiments, suggesting inability to carry out autotrophic denitrification. On the other hand, Pseudomonas aeruginosa reached 78% efficiency on acetate-only medium, suggesting a preference towards acetate as carbon source over bicarbonate. Environmental isolates presented an interesting potential of tolerating the potential inhibitory effects of sodium bicarbonate in the medium compared to Pseudomonas aeruginosa, which demonstrated inhibitory effects in this medium.